Direct comparison of nick-joining activity of the nucleic acid ligases from bacteriophage T4

The genome of bacteriophage T4 encodes three polynucleotide ligases, which seal the backbone of nucleic acids during infection of host bacteria. The T4Dnl (T4 DNA ligase) and two RNA ligases [T4Rnl1 (T4 RNA ligase 1) and T4Rnl2] join a diverse array of substrates, including nicks that are present in double-stranded nucleic acids, albeit with different efficiencies. To unravel the biochemical and functional relationship between these proteins, a systematic analysis of their substrate specificity was performed using recombinant proteins. The ability of each protein to ligate 20 bp double-stranded oligonucleotides containing a single-strand break was determined. Between 4 and 37 °C, all proteins ligated substrates containing various combinations of DNA and RNA. The RNA ligases ligated a more diverse set of substrates than T4Dnl and, generally, T4Rnl1 had 50-1000-fold lower activity than T4Rnl2. In assays using identical conditions, optimal ligation of all substrates was at pH 8 for T4Dnl and T4Rnl1 and pH 7 for T4Rnl2, demonstrating that the protein dictates the pH optimum for ligation. All proteins ligated a substrate containing DNA as the unbroken strand, with the nucleotides at the nick of the broken strand being RNA at the 3'-hydroxy group and DNA at the 5'-phosphate. Since this RNA-DNA hybrid was joined at a similar maximal rate by T4Dnl and T4Rnl2 at 37 °C, we consider the possibility that this could be an unexpected physiological substrate used during some pathways of 'DNA repair'

Immobilized DNA hairpins for assay of sequential breaking and joining of DNA backbones

Immobilized DNA hairpins are exploited in a novel approach to assay DNA ligases and nucleases. A fundamental characteristic of the assay is that a fluorophore at the remote terminus of the hairpin reports on the integrity of the DNA backbone. The functionality of the protocol is confirmed using ATP- and NAD+-dependent DNA ligases and the nicking enzyme N.BbvCIA. The assay format is amenable to high-throughput analysis and quantitation of enzyme activity, and it is shown to be in excellent agreement with the more laborious electrophoretic approaches that are widely used for such analyses. Significantly, the assay is used to demonstrate sequential breaking and rejoining of a specific nucleic acid. Thus, a simple platform for biochemically innovative studies of pathways in cellular nucleic acid metabolism is demonstrated